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Novel Kinetic Solution-Based Separation Approaches for Small Molecule Drug Discovery

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dc.contributor.advisorKrylov, Sergey N.
dc.creatorBao, Jiayin
dc.date.accessioned2017-07-27T12:46:50Z
dc.date.available2017-07-27T12:46:50Z
dc.date.copyright2017-01-04
dc.date.issued2017-07-27
dc.date.updated2017-07-27T12:46:50Z
dc.degree.disciplineChemistry
dc.degree.levelDoctoral
dc.degree.namePhD - Doctor of Philosophy
dc.description.abstractThe modern pharmaceutical industry has achieved remarkable successes in medicinal chemistry. However, many diseases are incurable due to the difficulty of finding new drugs. De novo drug discovery contains two steps: the primary screening focuses on selecting protein (target) binding drug (ligand); the secondary screening concentrates on calculating kinetic binding parameters of target-ligand complex. Conventional methods for the primary screening are typically surface-based, which suffer intensely from nonspecific interactions; the existing methods for secondary screening are either affinity-based or require surface immobilization, both cannot accurately calculate kinetic binding parameters. Hence, this research focuses on the development of the solution-based kinetic platform that facilitates both primary and secondary screenings. We combined kinetic capillary electrophoresis (KCE) with DNA-encoded ligand (DEL) technology to build a solution-based platform for primary screening of ligands. KCE offers high partitioning efficiency but requires the knowledge of electrophoretic mobility of target-ligand complex, and thus, we developed a mathematical model to predict electrophoretic mobility of target-DEL complex. This model was tested by using the targets interacted with 18 artificial DELs that contain various combinations of dsDNA and ssDNA regions, together with 2 DELs manufactured by GlaxoSmithKline. The results confirmed the precision, accuracy, and ruggedness of our model. This model will facilitate the reliable use of KCE-DEL based primary screening. Next, we developed a kinetic size-exclusion chromatography-mass spectrometry (KSEC-MS) as the label-free solution-based platform for calculating kinetic binding parameters of target-ligand interactions in secondary screening. KSEC-MS employs size-exclusion chromatography to separate small molecule ligand from protein target-ligand complex without immobilization and mass spectrometry to detect small molecule without a label. The rate constants of complex formation and dissociation are calculated from the temporal ligand concentration profile. Methods of KSEC-MS have been developed by using 2 proteins with the corresponding drugs. The resulted kinetic and affinity binding parameters were validated, which confirmed the precision and accuracy of KSEC-MS. We foresee that the KSEC-MS will become a universal approach for the kinetic analysis of target-ligand interactions in secondary screening.
dc.identifier.urihttp://hdl.handle.net/10315/33488
dc.language.isoen
dc.rightsAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.
dc.subjectPharmaceutical sciences
dc.subject.keywordsAnalytical chemistry
dc.subject.keywordsBiomolecular interactions
dc.subject.keywordsProtein-small molecule interactions
dc.subject.keywordsDrug screening
dc.subject.keywordsTarget-ligand interactions
dc.subject.keywordsLabel-free drug selections
dc.subject.keywordsSolution based-kinetic methods
dc.subject.keywordsKSEC-MS
dc.subject.keywordsKCE-MS
dc.subject.keywordsKCE
dc.subject.keywordsMass spectrometry
dc.subject.keywordsKinetic size-exclusion chromatography
dc.subject.keywordsDNA-encoded ligand
dc.subject.keywordsDEL
dc.titleNovel Kinetic Solution-Based Separation Approaches for Small Molecule Drug Discovery
dc.typeElectronic Thesis or Dissertation

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